Molecular biology tools for algal engineering

ABSTRACT

The present invention provides compositions and methods for the genetic manipulation of Algal cells. The compositions and methods allow enhanced transfer of genetic material into Algal cells and the cloning and selection of genetically modified cells. Expression of proteins encoded by the genetic material will be enhanced by the methods and compositions of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/138,388filed Apr. 26, 2016, which is a divisional of U.S. application Ser. No.14/670,309 filed Mar. 26, 2015 (now abandoned); which is a divisional ofU.S. application Ser. No. 13/584,615 filed Aug. 13, 2012 (now U.S. Pat.No. 9,018,013); which claims the benefit of priority of U.S. ProvisionalApplication No. 61/523,138, filed Aug. 12, 2011 which are all herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to the field of algal cell biology.More specifically, it relates to compositions and methods of cloning andexpressing nucleic acids molecules in Algae.

BACKGROUND

Algal biotechnology has strong potential to solve pressing challengesrelating to the availability of food, renewable energy and climatechange. Yet the tools for algal biotechnology do not currently allow forswift adoption of the organisms for synthetic biology applications. Mainareas of research that need attention are: high level transgeneexpression, targeted integration, homologous recombination, eliminationof silencing, ability to efficiently deliver large sections ofheterologous DNA as well as robust tools for production organisms.Methods and compositions delineated below seek to address thesechallenges thru commercialization of advanced synthetic biology toolkitsfor algal hosts.

SUMMARY

The present invention provides nucleic acids, vectors, plasmids, hostcells, buffers and methods for cloning genes and expressing proteins inalgal cells. These methods and compositions provide a collection oftools for manipulating the genome of an algal cell and cloning andselection of desired strains so that a high level expression of genes ofinterest may be obtained. Some embodiments provide for a composition fortransformation of an algal cell with DNA the composition comprising oneor more sugars, and a biological buffer. In particular embodiments theone or more sugars are selected from the group consisting of sucrose,fructose, maltose, trehalose, sorbitol, maltitol, erythrytol, mannitol,xylose, raffilose and lactose. In further embodiments the one or moresugars are present at a total concentration of from 40 mM to 100 mM. Forother embodiments the biological buffer is selected from the groupconsisting of Bis-Tris Propane, TRIS, AMPD, TABS, AMPSO, CHES and CAPSOand in other embodiments the concentration of the biological buffer isfrom 5 mM to 100 mM and may have a pH from 8 to 10. Some embodimentsprovide for an isolated nucleic acid which exhibits promoter activity inan algal cell. Another embodiment may be an algal cell comprising anisolated nucleic acid sequence which exhibits promoter activity. Furtherembodiments may be methods for performing homologous recombination in analgal cell comprising co-transforming the algal cell with a protein thatenhances homologous recombination. Other embodiments may provide for acyanobacteria derived from cyanobacteria strain BC104 comprising one ormore resistance markers and one or more promoters. A further embodimentmay be a plasmid comprising an origin of replication and plasmidmaintenance regions derived from pANL. Another embodiment may be analgal cell large capacity vector capable of replicating in Chlorella. Afurther embodiment may be an algal cell comprising an isolated nucleicacid which encodes an RNA polymerase under the control of an induciblepromoter the isolated nucleic acid further comprising a reporter geneunder control by the same inducible promoter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the yield of transformants when different sugars are usedin the transformation buffer.

FIG. 2 shows the transformation efficiency when using the optimizedtransformation buffer at different pH.

FIG. 3 shows the transformation efficiency when using the optimizedtransformation buffer with the Bio-Rad Gene Pulser® II and Neon®electroporation devices.

DETAILED DESCRIPTION

Algae or algal cell, refer to plants or cells belonging to the subphylumAlgae of the phylum Thallophyta. The algae are unicellular,photosynthetic, oxygenic algae and are non-parasitic plants withoutroots, stems or leaves; they contain chlorophyll and have a greatvariety in size, from microscopic to large seaweeds. Green algae,belonging to Eukaryota-Viridiplantae-Chlorophyta-Chlorophyceae, can beused. Blue-green, red, or brown algae may also be used. Exemplary algaefor which the methods and reagents described herein may be used includethose of the genus Chlamydomonas and the genus Chlorella.

Cyanobacteria are photosynthetic bacteria which require light, inorganicelements, nitrogen sources, water and a carbon source, generally CO₂, tometabolize and grow. Cyanobacteria are photosynthetic prokaryotes whichcarry out oxygenic photosynthesis. The main product of the metabolicpathway of Cyanobacteria during aerobic conditions is oxygen andcarbohydrates. Exemplary cyanobacteria include those found in DonaldBryant, The Molecular Biology of Cyanobacteria, published by KluwerAcademic Publishers (1994). Representative examples includeSynechococcus such as Synechococcus lividus and Synechococcus elongatus;and Synechocystis such as Synechocystis minervae, such as SynchocystisSp PCC 6803.

Nucleic acid: As used herein, a nucleic acid is a sequence of contiguousnucleotides (riboNTPs, dNTPs or ddNTPs, or combinations thereof) of anylength, which may encode a full-length polypeptide or a fragment of anylength thereof, or which may be non-coding. As used herein, the terms“nucleic acid molecule” and “polynucleotide” may be usedinterchangeably.

Transformation is a process for introducing heterologous DNA into aplant cell, plant tissue, or plant. Transformed plant cells, planttissue, or plants are understood to encompass not only the end productof a transformation process, but also transgenic progeny thereof.

Transformed, transgenic, and recombinant refer to a host organism suchas a bacterium or a plant into which a heterologous nucleic acidmolecule has been introduced. The nucleic acid molecule can be stablyintegrated into the genome of the host or the nucleic acid molecule canalso be present as an extrachromosomal molecule. Such anextrachromosomal molecule can be auto-replicating. Transformed cells,tissues, or plants are understood to encompass not only the end productof a transformation process, but also transgenic progeny thereof.

Promoter: As used herein, a promoter is an example of a transcriptionalregulatory sequence, and is specifically a nucleic acid sequencegenerally described as the 5′-region of a gene located proximal to thestart codon. The transcription of an adjacent nucleic acid segment isinitiated at the promoter region. A repressible promoter's rate oftranscription decreases in response to a repressing agent. An induciblepromoter's rate of transcription increases in response to an inducingagent. A constitutive promoter's rate of transcription is notspecifically regulated, though it can vary under the influence ofgeneral metabolic conditions.

A gene that is codon-optimized for expression in an organism is a genewhose nucleotide sequence has been altered with respect to the originalnucleotide sequence, such that one or more codons of the nucleotidesequence has been changed to a different codon that encodes the sameamino acid, in which the new codon is used more frequently in genes ofthe organism of interest than the original codon. The degeneracy of thegenetic code provides that all amino acids except form methionine andtryptophan are encoded by more than one codon. For example, arginine,leucine, and serine are encoded by different six different codons;glycine, alanine, valine, threonine, and proline are encoded by fourdifferent codons. Many organisms use certain codons to encode aparticular amino acid more frequently than others. Thus, for adequate oroptimal levels of expression of an encoded protein, a gene may becodon-optimized to change one or more codons to new codons (preferredcodons) that are among those used more frequently in the genes of thehost organism (referred to as the codon preference of the organism). Asused herein, a codon-optimized gene or nucleic acid molecule of theinvention need not have every codon altered to conform to the codonpreference of the intended host organism, nor is it required thataltered codons of a codon-optimized gene or nucleic acid molecule bechanged to the most prevalent codon used by the organism of interest.For example, a codon-optimized gene may have one or more codons changedto codons that are used more frequently that the original codon(s),whether or not they are used most frequently in the organism to encode aparticular amino acid.

Algal Transformation Reagent

The transformation efficiency in Chlamydomonas using conventionaltransformation reagents and procedures is extremely low and may limitthe use of this organism for high throughput applications such astesting libraries of evolved proteins or libraries of cDNAs from otherorganisms. To address this issue we have developed an optimizedtransformation buffer and corresponding optimized electroporationconditions using the Neon® (Life Technologies, Carlsbad, Calif.) andBio-Rad Genepulse® II (BioRad, Hercules, Calif.) electroporationapparatus.

The transformation buffer may be comprised of one or more sugars and abuffer salt to regulate pH. Suitable sugars include but are not limitedto sucrose, fructose, maltose, trehalose, sorbitol, maltitol,erythrytol, mannitol, xylose, raffilose and lactose. The total sugarconcentration may be from 40 mM to 100 mM, 50 mM to 100 mM, 60 mM to 100mM, 70 mM to 100 mM, 40 mM to 90 mM, 40 mM to 80 mM, 40 mM to 70 mM or40 mM to 60 mM. When more than one sugar is used the concentration ofany one sugar may be from 10 mM to 100 mM.

The pH of the transformation buffer may be from pH 7 to pH 10, pH 8 topH 10, pH 9 to pH 10, pH 7 to pH 9 or pH 7 to pH 8. A number ofBiological Buffers suitable for this pH range are available fromSigma-Aldrich (St. Louis, Mo.) including, but not limited to, Bis-TrisPropane, TRIS, AMPD, TABS, AMPSO, CHES and CAPSO. The concentration ofthe buffer may be from 5 mM to 100 mM, 5 mM to 90 mM, 5 mM to 80 mM, 5mM to 70 mM, 5 mM to 60 mM, 5 mM to 50 mM, 10 mM to 100 mM, 20 mM to 100mM, 30 mM to 100 mM, 40 mM to 100 mM or 50 mM to 100 mM.

Identification of Strong Promoters for Eukaryotic Algae.

Large DNA viruses that infect Chlorella have been used in the past as asource of strong promoters that have been applied to biotechnologicalapplications in plants and bacteria. With the emergence of algalbiotechnology, the isolation of these promoters for algal vectordevelopment holds even greater promise. Previously, viral genomes werepanned for highly active promoters in a high-throughput manner bygenerating a library of randomly sheared fragments of the >300 kb viralgenomes. Several strong promoters were isolated that function in plants.Since there are many different Chlorella isolates and many large DNAalgal viruses, the possibility of isolating a wide range of promotersthat have a myriad of useful qualities exist. These large DNA virusesemploy a wide range of DNA methylation patterns suggesting that they mayhave varying degrees of resistance to gene silencing due to DNAmethylation when used in vectors.

To identify the viral promoters with varying and useful qualities, viralgenomes may be sheared or cleaved by a unique Chlorella virus encodedrestriction endonuclease. Each fragment may then be cloned into abidireactional reporter vector and screened. Ideally, reporters that areamenable to FACS sorting such as GFP will be used. Strong promoters willbe isolated and validated with additional reporter genes. Candidatepromoters will also be evaluated for maintenance of expression as afunction of culture time to address silencing.

Development of Genetic Tools for Algal Production Strain—Chlorella.

Chlorella is a widely used platform organism in algal biotechnology withwell established mass culture. It is typically grown for consumption asa health supplement or animal feed due to its high protein content aswell as high levels of polyunsaturated fatty acids. Chlorella is alsonow increasingly used for biofuels research due to its high lipidcontent as well as ability for many strains to grow under heterotrophicconditions.

Initial reports show that genetic manipulation of Chlorella is feasiblehowever the tools for this organism are in their infancy.

Functional chlorella viral promoter elements will be evaluated in aselected set of Chlorella strains such as: C. vulgaris, C.protothecoides, C. pyrenoidosa, C. ellipsoidea. Reporter gene expressionfrom the promoters may be tested together with evaluation of commonresistance markers such as hygromycin and their optimization for use inChlorella. Development of additional resistance markers with massculture potential such as glyphosate resistance may be of value as well.Lastly feasibility of performing targeted integration in Chlorella willbe established.

Enable Homologous Recombination in Eukaryotic Algae.

Targeted integration can alleviate the need for screening large numbersof clones and can generally result in more uniform expression levelsacross different clones however it does not address the inability todisrupt genes in green algae in a targeted fashion. The need stillexists for homologous recombination driven gene disruption to enablequick generation of e.g. deletion mutants or promoter replacements inthe nuclear genome.

Homologous recombination between the organism's genome and introducedDNA has been shown to occur in many organisms including yeast andmammalian cells, but remains difficult in photosynthetic organisms.Nevertheless homologous recombination does occur in Chlamydomonasnuclear genome albeit at a low frequency. Specifically, the NIT8 locushas been used to deliver a selectable marker, CRY1-1 that confersemetine resistance, via homologous recombination. Disruption of NIT8results in chlorate resistance thus allowing for selection oftransformants that disrupted this locus. Following selection with bothchlorate and emetine, a construct containing 8.2 kb of homology anddesigned to disrupt NIT8 with CRY1-1 was found to correctly disrupt thelocus in only 1/2000 transformants. Strategies to increase the rate ofhomologous recombination may include: co-transformation with RecA orother proteins such as eukaryotic Rad51 homologs, evaluation of factorsthat affect recombination efficiency (e.g. DNA amount, length ofhomology, electroporation conditions, cell culture conditions), use ofDNA single or double strand break agents or irradiation, as well aseffects of cell cycle.

Highly efficient homologous recombination does occur in theChlamydomonas chloroplast. Characterization of genes that facilitatethis process in the chloroplast given its' small size appears feasiblewith consequent expression of them in the nucleus and re-evaluation ofefficiency of nuclear homologous recombination.

Improvement of Genetic Tools for a Cyanobacterial Production Strain.

Cyanobacteria have several traits that make them attractive productionhosts. They have a wide range of metabolic capabilities while havinglittle nutritional requirements. Some cyanobacteria not only fix carbondioxide but also fix nitrogen reducing the need for fertilizer. They cantolerate high pH, high light intensity (including protection from UVlight) and often high salt—traits that offer crop protection. Manycyanobacteria also produce mucilaginous envelope that protects themagainst predators and/or desiccation. Most importantly, cyanobacteriabeing prokaryotic are easy to manipulate genetically and offeradvantages of cistronic expression as well as small genomes that can bemore easily characterized—important traits for synthetic biology hosts.

Strain BC104 (or BL0902) was isolated in Imperial Valley, Calif. anddeveloped as a production strain due to presence of many favorabletraits. BC104 belongs to the Leptolyngbya sp., is filamentous, showsrobust growth in 20-40° C. temperature range, can tolerate high pH (pH11) and urea (used for predator control), grows in up to 0.5M salt (seawater concentration) and tolerates high solar irradiance. The growthrate of BC104 exceeds that of Spirulina in laboratory culture and is onpar with Spirulina outdoors with excellent culture stability. BC104 canalso be harvested using similar screening methods used for Spirulina.BC104 has been shown to accumulate >25% fatty acids/dry cell weightfollowing conversion to FAME. In addition to these desirable qualities,BC104 is amenable to transformation that is reliable, efficient, andstable. Transformation may be demonstrated by the introduction of aplasmid that encodes yemGFP on RSF1010 broad host range origin plasmidby conjugation.

Whereas the production traits of BC104 have been well characterized, thegenetic tool box for this strain is still rather small. Furtherdevelopment of the tools will require a set of robust resistancemarkers, evaluation of additional promoters, demonstration of expressionof non-reporter genes that are relevant biotechnologically as well asdemonstration of gene knockouts via homologous recombination. Moreover,the organism's genome will be sequenced and annotated.

Development of High Capacity Gene Transfer System for Cyanobacteria.

There are several methods for DNA delivery into cyanobacteria, the mostprevalent being conjugation and natural transformation. Naturaltransformation has the advantage of ease of use but is limited to fewmodel organisms such as Synechococcus elongatus and Synechocystis.Conjugation is more widely used and has many advantages such as highefficiency of DNA transfer, low species selectivity and capacity totransfer very large DNA segments with limits typically imposed by therecipient organism rather than transfer capacity. Conjugation from E.coli has been successfully used to deliver DNA to many cyanobacterialspecies such as: Synechococcus elongatus PCC7942, Anabaena PCC7120,Nostoc punctiforme ATCC 29133, Cyanothece sp. ATCC 51142, Synechococcussp. WH8102, Chroococcidiopsis sp. Tolypothrix sp. PCC7601.

Given robust high capacity DNA transfer mechanisms, next step isestablishment of a universal plasmid or small subset of plasmids withbroad host range specificity. A good candidate for origin of replicationis based on RSF1010 plasmids (oriV, mob, rep) which has been shown toreplicate, albeit with poor efficiency in some cases, in distant speciesof cyanobacteria: Synechococcus elongatus PCC7942, SynechocystisPCC6803, Synechocystis PCC6714, Anabaena PCC7120, Cyanothece sp. ATCC51142, Leptolyngbya sp. BL0902 (BC104). Improvement of host rangespecificity in addition to replication efficiency in cyanobacteria canbe done by sequential mutagenesis and selection in a group ofcyanobacterial strains of interest.

RSF1010 appears to have poor stability in Synechococcus elongatusPCC7942. Development of the large endogenous plasmid pANL for highcapacity cloning may prove to be a more short term solution for thisplatform organism. pANL is 46 kb in length, 53% GC content and encodes58 orfs. There are 4 structural and functional regions that have beencharacterized: the replication origin region, signal transductionregion, plasmid maintenance region (containing a toxin-antitoxinaddition cassette) and sulfur-regulated region. Both replication originand plasmid maintenance regions are required for persistance of pANL inthe cells.

To enable high capacity cloning in Synechococcus elongatus, thereplication origin and plasmid maintenance origins from pANL will firstbe minimized in size while maintaining functionality and then combinedwith yeast elements to allow high order assembly as well as elements toenable conjugation from E. coli if necessary. The hybrid vector will beevaluated for stability as well as DNA carrying capacity.

Development of Viral Gene Transfer System for High Capacity Cloning inGreen Algae.

An important application of algae genetic engineering tools would be thedevelopment of an easily useable and reliable viral vector for genedelivery and integration. A wide variety of viral vectors exist formammalian cell systems and agrobacterium provide an extremely versatileand powerful system in plants. While recombinant manipulation andsubsequent transformation of algae hosts with the large DNA algalviruses has proven elusive to date, the potential for developing theseviruses as vectors is strong.

Initial efforts in the development of large capacity viral deliverysystem for eukaryotic algae may focus on large chlorella viruses such asthe PBCV-1 virus and their host Chlorella NC64A with key aspect ofunderstanding and controlling lysis vs lysogeny of the virus. Severalapproaches may be necessary to identify the lysis genes such astranscriptome analysis of early, middle and late expressed genes duringinfection in concert with bioinformatics analysis of gene candidates. Ahigh throughput approach of transposon mutagenesis of viral particlesand screening for infection but lack of lysis may be tried.Understanding the mechanisms of lysis may not only be valuable from thestandpoint of engineering a gene delivery method but also fromdiscovering methods to engineer inducible lysis for product separationapproaches.

In addition to identification of lysis genes, the capacity to introduceand carry exogenous DNA will need to be evaluated with concomitantgenome streamlining of the virus to increase that capacity.

Development of Chlamydomonas T7 RNA Pol/T7 Promoter ChloroplastExpression Platform.

T7 RNA polymerase is an RNA polymerase from T7 bacteriophage withextremely high specificity towards the T7 promoter, high processivityand low error rate. T7 RNA polymerase is commonly used in E. coliexpression platforms (e.g. BL21 DE3) and has been successfully appliedto drive expression from the T7 promoter in several organisms including:S. cerevisiae mitochondria, E. coli, Bacillus megaterium andPseudomonas. Recently the Voigt lab at UCSF has developed variants of T7RNA polymerases with altered processivity and specificity as well as asuite of T7 promoters of different strength.

Given the demonstration that T7 RNA polymerase system can be used inyeast mitochondria as well as the prokaryotic nature of chloroplastexpression machinery, it should be feasible to apply the T7 RNApolymerase/T7 promoter system to express genes in the chloroplast. Bothwild-type T7 RNA polymerase as well as the low processivity (Voigt lab)T7 RNA polymerase (codon optimized for chloroplast expression) will beevaluated. The polymerase may be placed under control of an induciblepromoter to enable regulated levels of expression. A reporter gene suchas GFP may be placed under control of the T7 promoter and the expressionof GFP following induction of T7 RNA polymerase will be evaluated.

Example 1: Transformation Yield with Transformation Buffers HavingDifferent Sugar Compositions

Wild type Chlamydomonas reinhardtii cells were washed twice with 2.5 mlof an electroporation buffer comprising 40 mM of the selected sugar, and10 mm Tris adjusted to a pH of 7.4. After washing, the cells wereresuspended in the electroporation buffer at a concentration of 2×10⁸cells/ml. For each electroporation reaction, 250 μl cells were mixedwith 2 μg of V1-Gus-ScaI linear DNA and incubated at 4° C. for 5 min.Immediately before electroporation the reaction mixture was transferredto a pre-chilled cuvette and then electroporation performed in a Bio-RadGenepulser® II apparatus with settings of 500V, 50 mF and 800 W. Thereactions were set on the bench for 15 min for resting and thentransferred into 10 ml of TAP media with 40 mM sucrose to recover overnight with light. Transformation efficiency was determined by plating1/100 of each reaction. The results are shown in FIG. 1.

Example 2: Transformation Yield with Transformation Buffers HavingDifferent pH

Wild type Chlamydomonas reinhardtii cells were washed twice with 2.5 mlof an electroporation buffer comprising 40 mM sucrose, 10 mM sorbitoland 10 mm CHES adjusted to a pH of between 8.5 and 9.5. After washing,the cells were resuspended in the electroporation buffer at aconcentration of 2×10⁸ cells/ml. For each electroporation reaction, 250μl cells were mixed with 2 μg of V1-Gus-ScaI linear DNA and incubated at4° C. for 5 min. Immediately before electroporation the reaction mixturewas transferred to pre-chilled cuvettes and then electroporationperformed in a Bio-Rad Genepulser® II apparatus with settings of 500V,50 mF and 800 W. The reactions were set on the bench for 15 min forresting and then transferred into 10 ml of TAP media with 40 mM sucroseto recover over night with light. Transformation efficiency wasdetermined by plating 1/100 of each reaction. The results are shown inFIG. 2.

Example 3: Transformation Yield with Different Vectors andElectroporation Apparatus

Wild type Chlamydomonas reinhardtii cells were washed twice with 2.5 mlof an electroporation buffer comprising 40 mM sucrose, 10 mM sorbitoland 10 mm CHES adjusted to a pH of 9.25. After washing, the cells wereresuspended in the electroporation buffer at a concentration of 2×10⁸cells/ml. For each electroporation reaction, 2500 cells were mixed with2 μg of DNA and incubated at 4° C. for 5 min. Immediately beforeelectroporation the reaction mixture was transferred to pre-chilledcuvettes and then electroporation performed in a Bio-Rad Genepulser® IIor Neon® apparatus with settings of 500V, 50 mF and 800 W. The reactionswere set on the bench for 15 min for resting and then transferred into10 ml of TAP media with 40 mM sucrose to recover over night with light.Transformation efficiency was determined by plating 1/100 of eachreaction. The results are shown in FIG. 3.

1. A composition for transformation of an Algal cell with DNAcomprising: (a) one or more sugars, and (b) a biological buffer.
 2. Thecomposition of claim 1, wherein the one or more sugars are selected fromthe group consisting of sucrose, fructose, maltose, trehalose, sorbitol,maltitol, erythrytol, mannitol, xylose, raffilose and lactose.
 3. Thecomposition of claim 2, wherein the one or more sugars are sucrose andsorbitol.
 4. (canceled)
 5. The composition of claim 1, wherein thebiological buffer is selected from the group consisting of Bis-TrisPropane, TRIS, AMPD, TABS, AMPSO, CHES and CAPSO.
 6. (canceled)
 7. Thecomposition of claim 1, wherein the pH is from 8 to
 10. 8. (canceled) 9.The composition of claim 1, further comprising an algal cell. 10.(canceled)
 11. A method for transformation of DNA into an algal cellcomprising: (a) suspending the algal cells in a buffer, (b) adding DNAto be transformed to the algal cell suspension, and (c) applying anelectric pulse to the DNA Algal cell suspension thereby transforming thealgal cell with the DNA.
 12. The method of claim 11, wherein the buffercomprises one or more sugars and a buffer salt.
 13. The method of claim12, wherein the one or more sugars are selected from the groupconsisting of sucrose, fructose, maltose, trehalose, sorbitol, maltitol,erythrytol, mannitol, xylose, raffilose and lactose.
 14. The method ofclaim 12, wherein the one or more sugars are sucrose and sorbitol. 15.The method of 11, wherein the pH of the buffer is from pH 8 to pH 10.16. The method of claim 15, wherein the pH of the buffer is from pH 9 topH
 10. 17.-22. (canceled)
 23. A method for performing homologousrecombination in an algal cell comprising co-transforming the algal cellwith a protein that enhances homologous recombination. 24.-27.(canceled)
 28. The method of claim 23, wherein homologous recombinationoccurs in the nucleus of the algal cell.
 29. The method of claim 23,wherein DNA and RecA protein are introduced into the algal cell.
 30. Themethod of claim 23, wherein the algal cell is a eukaryotic cell.